The general goal of this work is to investigate the association of mRNA with the cytoskeleton in certain mammalian cells, such as HeLa. In particular, we wish to find out if this association has a role in mRNA translation, and if it occurs through mRNP proteins alone, or if ribosomes also bind to the cytoskeleton, as suggested by our preliminary results. mRNP proteins are defined here as proteins in direct contact with mRNA in intact cells and isolated ribonucleoproteins by the criterion of crosslinking to mRNA by irradiation with ultraviolet light. The objectives can be divided into three parts. The first is devoted to obtaining additional information about some of the mRNP proteins. Specifically, we shall investigate their relationship to known translation factors such a eIF-4B and cap binding proteins, further investigate the role of mRNP cap-associated proteins in initiation of translation, and investigate the association of cap-associated and other mRNP proteins with mRNA in intact cells. These experiments will utilize ultraviolet light-induced crosslinking, partial peptide maping, immunoaffinity chromatography with monoclonal antibodies to cap binding proteins, and reversible inhibition of translation initiation by heat shock. The second part will deal with improving procedures for the isolation of cytoskeletons with attached mRNA and ribosomes. Two approaches will be tried: altering the cell lysis conditions and fixation with a reversible protein-protein crosslinking agent. The third part will consist of investigating the association of mRNP proteins and ribosomes with the cytoskeleton. RNAase digestions will be done to determine whether the mRNP proteins are attached to the cytoskeleton through RNA, or whether they bind directly to proteinaceous elements of the cytoskeleton. Our preliminary results suggest that some mRNP proteins bind to ribosomal RNA as well as to mRNA. This observation will be further explored as a possible mechanism for binding of both mRNA and ribosomes to the cytoskeleton. Intact cells will be irradiated, and the proteins crosslinked to RNA in the various ribosomal particles attached and not attached to the cytoskeleton will be analyzed. The crosslinked rRNA-protein complexes will be isolated by hybridization to cloned ribosomal DNA immobilized on cellulose.